Clostridium difficile causes a potentially fatal intestinal disease that is increasing in incidence and severity. These infections are often chronic and incredibly difficult to eradicate. Though this organism presents an enormous public health burden, little is understood about how C. difficile evades host defenses to colonize the human intestine. In order to persist, the bacteria must cope with a continuous onslaught by host defenses. The long-term goal of this project is to reveal how C. difficile evades host innate immunity with the intent of opening new avenues for treatment. The specific objective of this application is to define the mechanisms C. difficile employs to resist cationic antimicrobial peptides (CAMPs) produced by the host. These peptides play a critical role in host innate defenses, preventing the growth and spread of bacteria. Preliminary data obtained thus far indicate that C. difficile can respond and adapt to the presence of CAMPs. I hypothesize that C. difficile has evolved multiple resistance mechanisms that allow the bacterium to bypass host immune responses, such as CAMPs, allowing it to colonize and replicate in the human intestine. This proposal will identify the genetic mechanisms of CAMP resistance in C. difficile, identify and characterize the regulation of CAMP resistance mechanisms, and determine the role of these mechanisms in the virulence and colonization in vivo. CAMP resistance will be studied using traditional genetic and biochemical methods, along with new technologies, such as rapid, whole-genome sequencing. Successful completion of these aims will illuminate fundamental aspects of C. difficile infection and identify potential therapeutic targets for treatment of disease. Furthermore, this knowledge will better our understanding of how host-bacterial interactions in the intestine can lead to digestive diseases. The experiments outlined in this proposal will help me learn a wide range of molecular genetics, biochemistry, and animal research techniques I have not previously used, and also to master methods for use with this important hospital pathogen. Ultimately, this work will set the stage for my future research on C. difficile pathogenesis and provide the framework for my transition into an independent investigator position.